Water Soluble Cholesterol Derivatives

ABSTRACT

Four novel water soluble cholesterol derivative compounds are disclosed. These compounds have various applications in studies of membrane proteins, including drug screening and studies of receptor stability and folding. In one aspect the water soluble cholesterol derivatives disclosed may be used to replace cholesterol in micelle-solubilized membrane protein preparations.

REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/494,159, filed Jun. 29, 2009, which claims priority to U.S.Provisional Patent Application Ser. No. 61/082,114, filed on Jul. 18,2008, which are hereby incorporated herein by reference in theirentirety for all purposes.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Small BusinessInnovation Research (SBIR) Program grant number R43MH081379 awarded bythe National Institutes of Health. The government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates generally to water-soluble derivatives ofcholesterol and methods for their synthesis and use.

BACKGROUND OF THE INVENTION

In order to be fully functional, many cellular receptors and othermembrane proteins require interactions with cholesterol within the lipidbilayer. Therefore, when reconstituting these proteins for in vitrobiochemical and structural studies, it is desirable to mimic the nativebilayer as closely as possible. This can be accomplished by adding theappropriate lipids and cholesterol to micelle-solubilized membraneprotein preparations. One obstacle however, is that cholesterol isnotoriously difficult to solubilize under the micellar conditionstypically used for membrane protein solubilization. In the presentinvention, four novel water soluble cholesterol analogs are disclosedwhich have various applications in studies of membrane proteins.

SUMMARY OF THE INVENTION

We have prepared four water-soluble cholesterol derivatives depicted inthe Figures. FIG. 1A is Cholesterol-β-tetrasaccharide (1), FIG. 1B isCholesterol-α-tetrasaccharide (2), and FIG. 1C is Cholesterol-β-sulfatedmaltoside (3). FIG. 1D is Diosgenin-β-tetrasaccharide (4). In oneembodiment, the compounds are made by multi-step chemical synthesis fromcommercial cholesterol or diosgenin (other steroids are potentialsubstrates and are denoted as ‘cholesterol derivative’) and maltose.

In preferred aspects, the tetrasaccharide is prepared via selectivecoupling of two differentiated units of a protected maltose. For eachcompound a final coupling step between the cholesterol derivative andthe saccharide is required. Descriptions of alpha and beta aredescriptors of the anomeric linkage between the cholesterol derivativeand the first sugar. Each compound can be made on a scale of up to 5 gand they are soluble up to 5% w/v in water.

BRIEF DESCRIPTION OF THE DRAWING

The accompanying drawing, which is incorporated in and forms a part ofthis specification, illustrates embodiments of the invention and,together with the description, serves to explain the invention:

FIG. 1A shows the structure of Cholesterol-β-tetrasaccharide (1). FIG.1B shows the structure of Cholesterol-α-tetrasaccharide (2). FIG. 1Cshows the structure of Cholesterol-β-sulfated maltoside (3). FIG. 1Dshows the structure of Diosgenin-β-tetrasaccharide (4).

DETAILED DESCRIPTION OF THE INVENTION General

The present invention has many embodiments and relies on many patents,applications and other references for details known to those of the art.Therefore, when a patent, application, or other reference is cited orrepeated below, it should be understood that it is incorporated byreference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

DEFINITIONS

Cholesterol is a lipid compound with a molecular formula C₂₇H₄₆O,consisting of three structurally distinct regions: i) a rigid sterolring, ii) a flexible alkyl chain, together forming the hydrophobicportion of the molecule, and iii) 3β-hydroxyl moiety, which is the onlypolar group in cholesterol and gives the molecule its amphiphaticcharacter. Cholesterol is an essential component of cell membranes inall eukaryotes, with crucial roles in membrane organization, dynamics,function and sorting.

Cholesterol is found distributed non-randomly in domains and pools inbiological membranes, and has been reported to modulate conformation(and hence function) of integral membrane proteins, either through aspecific molecular interactions, or through alterations in the membranephysical properties induced by the presence of cholesterol.

An example of a membrane protein family regulated by cholesterol is theseven transmembrane domain G-protein coupled receptors (GPCRs). The GPCRfamily has over 800 members, representing approximately 2% of totalproteins encoded by the human genome. GPCRs have diverse signalingfunctions, mediating responses to both endogenous and exogenous ligandsvia recruitment and activation of heterotrimeric G-proteins to thecytoplasmic side of the cell membrane. GPCR-activated heterotrimericG-proteins in turn regulate a multitude of physiological processesranging from neurotransmission and cellular differentiation and growthto inflammatory and olfactory responses. Therefore, GPCRs representmajor targets for the development of new drugs.

The term liposome, as used in this application, refers to a vesicularstructure composed of a lipid bilayer separating an aqueous internalcompartment from the bulk aqueous phase. Liposomes can be composed ofnaturally-derived phospholipids with mixed lipid chains, or of puresurfactant components such as DOPE (dioleoylphosphatidylethanolamine).The term micelle, as used in this application, refers to closed lipidmonolayers with a fatty acid core and polar surface, or for an invertedmicelle, a polar core with fatty acids on the surface. Due to theirunique structure, both liposomes and micelles have been used in vitrostudies of protein function as well as drug delivery.

The term water-soluble cholesterol derivative, as used in thisapplication, refers to cholesterol analogs containing a water-solublehead group or moiety.

Water-Soluble Cholesterol Derivatives

Commercially available water-soluble cholesterol derivatives, such ascholesterol hemisuccinate and cholesterol sulfate, have been used ascholesterol substitutes in studies of membrane protein function andstructure. These cholesterol analogs contain anionic head groups, makingthem more suitable for micellar membrane protein preparations. However,cholesterol hemisuccinate and cholesterol sulfate remain difficult tosolubilize in detergent micelles, and their anionic head groups makethem not biochemically optimal for in vitro protein studies. Therefore,there remains a need for the development of water-soluble cholesterolderivatives that can be better utilized in biochemical and structuralstudies of membrane proteins.

To address this issue, we have synthesized novel, water-solublecholesterol derivatives that can be used to enhance the solubility andfunctionality of micelle-solubilized membrane proteins for biochemicaland structural studies. These compounds will likely be useful in otherapplications that benefit from abundant quantities of pure andproperly-folded membrane proteins (ie, GPCRs), including drug screeningand studies of receptor stability and folding.

In a preferred aspect, the compounds are made by multi-step chemicalsynthesis from commercial cholesterol or diosgenin (other steroids arepotential substrates and are denoted as ‘cholesterol derivative’) andmaltose.

In preferred aspects, the tetrasaccharide is prepared via selectivecoupling of two differentiated units of a protected maltose. For eachcompound a final coupling step between the cholesterol derivative andthe saccharide is required. Descriptions of alpha and beta aredescriptors of the anomeric linkage between the cholesterol derivativeand the first sugar. Each compound can be made on a scale of up to 5 gand they are soluble up to 5% w/v in water.

CONCLUSION

It is to be understood that the above description is intended to beillustrative and not restrictive. Many variations of the invention willbe apparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should be determined withreference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. All cited references,including patent and non-patent literature, are incorporated herewith byreference in their entireties for all purposes.

What is claimed is:
 1. Cholesterol-β-tetrasaccharide according toFormula I:


2. Cholesterol-α-tetrasaccharide according to Formula II:


3. Cholesterol-β-sulfated maltoside according to Formula III, wherein Xis selected from the group consisting of Me₃NH, NH₄, and Na:


4. Diosgenin-β-tetrasaccharide according to Formula IV:


5. A composition comprising the cholesterol-β-tetrasaccharide accordingto claim
 1. 6. A composition comprising thecholesterol-α-tetrasaccharide according to claim
 2. 7. A compositioncomprising the cholesterol-β-sulfated maltoside according to claim
 3. 8.A composition comprising the diosgenin-β-tetrasaccharide according toclaim
 4. 9. A method of synthesizing the cholesterol-β-tetrasaccharideaccording to claim 1, wherein the synthesis begins with materialscomprising cholesterol and maltose.
 10. A method of synthesizing thecholesterol-α-tetrasaccharide according to claim 2, wherein thesynthesis begins with materials comprising cholesterol and maltose. 11.A method of synthesizing the cholesterol-β-sulfated maltoside accordingto claim 3, wherein the synthesis begins with materials comprisingcholesterol and maltose.
 12. A method of synthesizing thediosgenin-β-tetrasaccharide according to claim 4, wherein the synthesisbegins with materials comprising diosgenin and maltose.
 13. A method ofreconstituting membrane proteins in a lipid bilayer, comprising:providing membrane protein; providing at least one cholesterolderivative selected from the group consisting of:

and mixing the membrane protein and the at least one cholesterolderivative to reconstitute membrane protein suitable for in vitrobiochemical and structural studies.
 14. The method according to claim13, wherein the membrane protein is a G-protein coupled receptor (GPCR).15. The method according to claim 13, wherein the membrane protein is atransmembrane protein.